1. Field of the Invention
The present invention relates to novel benzopyranyl heterocycle derivatives of the structural formula 1. It also relates to process for preparing the novel compounds and pharmaceutical compositions comprising the compounds as an active ingredient.
The present invention also relates to pharmaceutical use of the benzopyranyl heterocycle derivatives. In particular, the present invention is useful in the prevention, treatment of diseases related to xe2x80x9cischemia-reperfusionxe2x80x9d injury such as ischemic heart, brain, neuronal cells and retina, and suppression of lipid peroxidation. 
Wherein R1, R2, R3, R4, n and * are each defined in specification.
2. Description of the Prior Art
Ischemic heart diseases are usually caused by myocardial ischemia, when the oxygen supply is significantly decreased compared to the oxygen demand due to the imbalance between them [G. J. Grover, Can. J. Physiol. 75, 309 (1997); G. D. Lopaschuk et al. Science and Medicine 42 (1997)]. Myocardial ischemia triggers various pathophysiological changes progressively that will ultimately lead to irreversible myocardial injury, cell death and tissue necrosis. At a stage where the injury to the cells is reversible, this process can be prevented by early reperfusion of the myocardium. However, there is potential for xe2x80x9creperfusion-injuryxe2x80x9d to occur [D. J. Hearse, Medicographia 18, 22 (1996)].
To delay the ischemic cascade and to minimize the reperfusion-injury, the use of adenosine agonists, inhibitors of Naxe2x88x92xe2x80x94Kxe2x88x92 antiport, oxygen free radical scavengers and KATP (ATP sensitive potassium channel) openers are investigated as well as ACE (Angiotensin converting enzyme) inhibitors and calcium antagonists. In addition, global ischemia occurs during cardiac surgery or during heart storage prior to transplantation. Recent studies reported that the addition of KATP openers to a hyperkalemic cardioplegic solution, improved the recovery of postischemic contractile function after normothermic or short periods of hypothermic ischemia [D. J. Chambers, D. J. Hearse, Ann. Thoar. Surg.; 68, 1960 (1999)].
Both global and focal ischemia or hypoxia initiate progressive cellular changes by a reduction of oxygen, which lead to brain injury, cell death, and tissue necrosis [K. Nieber, Pharmacol. Ther. 82, 71 (1999)]. Even after blood flow is restored, xe2x80x9creperfusion-injuryxe2x80x9d can be occurred same as in the heart. In order to prevent the brain injury and minimize the alteration of neuronal function, progressive pathophysiological changes arose from ischemia-reperfusion must be prevented. For that purpose, the development of several neuroprotectives such as excitatory amino acid antagonists, anti-oxidants, adenosine agonists and KATP channel openers are being pursued. The use of those compounds as protectants or curatives for the organs related to xe2x80x9cischemia-reperfusion injuryxe2x80x9d such as retina and skeletal muscles besides heart and brain, is being investigated, too.
Damage or death of neuronal cells is known to be a main reason for various neurological disorders such as stroke, Alzheimer""s disease, Parkinson""s disease, etc. [G. J. Zoppo et al., Drugs 54, 9(1997); I. Sziraki et al., Neurosci. 85, 1101 (1998)]. Various factors including increases in iron concentration, reactive oxygen species, and oxidants within neurons are known to initiate neuronal cell damages [M. P. Mattson et al., Methods Cell Biol. 46, 187 (1995); Y. Goodman et al., Brain Res. 706, 328 (1996)].
An increase of oxygen radicals may induce a lipid peroxidation, and thus their formation results in the accumulation of oxidants in neuronal cell. The oxidants accumulated in cells are known to be responsible for cardiac infarction, dementia, and inflammatory diseases such as arthritis as well as acute and chronic injury of tissues and organs caused by ischemia-reperfusion.
Therefore, therapeutic approaches to minimize neuronal injury by oxidative stress and inhibit lipid peroxidation have been pursued, which may prevent or treat the deseases caused by the damage or death of neuronal cells. To date, anti-oxidants are reported to ameliorate the neuronal damage and death caused by an increase of iron concentration within neurons. Much effort has been continued to develop pharmaceutical drugs which are able to prevent neuronal damage by oxidative stress [Y. Zhang et al., J. Cereb. Blood Flow Metab. 13, 378 (1993)]. Diazoxide, a KATP channel opener, has been reported to reversibly oxidize flavoproteins in mitochondria, resulting in inhibition of the formation of oxygen free radicals, which may protect cell injury by oxidative stress [A. A. Starkov, Biosci, Rep. 17, 273 (1997); V. P. Skulachev, Q. Rev. Biophus. 29, 169 (1996)]. In addition, there are reports that KATP opening is related to the induction of anti-oxidant enzymes [S. Okubo et al., Mol. and cell Biochem, 196, 3 (1999)], and to decrease the release of excitatory amino acid [J-L Moreau, G. Huber, Brain Res., 31, 65 (1999)].
KATP is found in a variety of tissues including cardiac muscle, skeletal muscle, pancreatic (xcex2-cells, and the brain, which makes it attractive as a drug target. However, the same diversity poses a difficulty of finding tissue selective agents. Differently from conventional potassium channel openers, the benzopyranyl indole analogue represented by the following formula 2 and benzopyranyl cyanoguanidine compound (BMS-180448) represented by the following formula 3, have been reported to show modest antiischemic potency with excellent cardiac selectivity. Although the compound represented by formula 2 had all desirable features to serve as a lead compound, the synthesis of it presented a major challenge [K. S. Atwal et al., J. Med. Chem. 38, 3236 (1995); K. S. Atwal et al., J. Med. Chem. 40, 24 (1996); K. S. Atwal et al., Current Pharmaceutical Design, 2, 585 (1996)]. Also, the conventional compounds, which have cardioprotective potency without a significant lowering of blood pressure, still give the prospects for the development of a novel cardioprotectant. 
Therefore, by the coupling of benzopyranyl epoxide and heterocyclic amine compounds which have increased nucleophilicity compared to indoleamines, the benzopyranyl heterocycle derivatives represented by the formula 1, having superior cardioprotective activity from ischemia-reperfusion damage, are synthesized in high yields. The compounds also exhibit various pharmaceutical efficacies, including protection of neuronal cells and prevention of lipid peroxidation and thus can be useful in the prevention and treatment of various diseases related to ischemia-reperfusion damage such as protection of heart, neuronal cells, retina, brain injury, and organ preservation for storage, or inhibition of lipid peroxidation.
One of the objectives of the present invention is to provide novel benzopyranyl heterocycle derivatives of formula 1.
Another objective of the present invention is to provide process for the preparation of the benzopyranyl heterocycle derivatives.
Further objective of the present invention is to provide pharmaceutical use of the benzopyranyl heterocycle derivatives. In particular, the present invention provides the use of the benzopyranyl heterocycle derivatives for the protection of heart, brain, retina from ischemic injury or protection of organ for storage, and suppression of lipid peroxidation.
The present invention provides benzopyranyl heterocycle derivatives represented by the following formula 1 and their pharmaceutically acceptable salts. 
Wherein
n is 0 or 1;
R1 represents H, NO2, or NH2;
R2 represents OH, or O(Cxe2x95x90O)Ra; and Ra represents H; straight or branched alkyl group of C1-C4; or aryl group;
R3 represents H, C(xe2x95x90O)ORa, CH2ORa, or C(xe2x95x90O)NRa2; and Ra is defined as above;
or R2 and R3 are connected to form lactone ring 
R4 represents H, halogen, OH, or ORa; and Ra is defined as above;
* represents the chiral center;
and single or double bond exists at 2,3-position of heterocycle.
In the formula 1, more preferably
n is 0 or 1;
R1 represents NO2 or NH2;
R2 represents OH;
R3 represents C(xe2x95x90O)ORa, or C(xe2x95x90O)NRa2; and Ra represents H; or straight or branched alkyl group of C1-C4;
or R2 and R3 are connected to form lactone ring 
R4 represents H, halogen, OH, or OCH3.
* represents the chiral center;
and single or double bond exists at 2,3-position of heterocycle.
The present invention includes all the solvates and hydrates which can be prepared from benzopyranyl heterocycle derivatives of formula 1 in addition to benzopyranyl heterocycle derivatives of formula 1 and their pharmaceutically acceptable salts.
The present invention includes all the separate stereochemical isomers, i.e. diastereomerically pure or enantiomerically pure compounds which have one or more chiral centers at 2, 3, 4 and 2xe2x80x2-positions, in addition to the racemic mixtures or diastereomer mixtures of benzopyranyl heterocycle derivatives of formula 1. In case of having four chiral centers at 2, 3, 4 and 2xe2x80x2-positions, the 3,4-dihydro benzopyran heterocycle derivatives according to the present invention are represented by the optical isomers such as (I1), (I2), (I3), (I4), (I5) (I6), (I7) and (I8) (See the following formula 4). 
Wherein R1, R2, R3, R4 and n are defined as above.
In particular, the preferable compounds of the present invention are:
1) 1-[(2S, 3R, 4S)-3,4-dihydro-2-dimethoxymethyl-3-hydroxy-2-methyl-6-nitro-2H-1-benzopyran-4-yl]-(2R)-2,3-dihydro-1H-indole-2-carboxylic acid ethyl ester;
2) 1-[(2S, 3R, 4S)-3,4-dihydro-2-dimethoxymethyl-3-hydroxy-2-methyl-6-nitro-2H-1-benzopyran-4-yl]-(2S)-2,3-dihydro-1H-indole-2-carboxylic acid ethyl ester;
3) 1-[(2S, 3S, 4R)-3,4-dihydro-2-dimethoxymethyl-3-hydroxy-2-methyl-6-nitro-2H-1-benzopyran-4-yl]-(2R)-2,3-dihydro-1H-indole-2-carboxylic acid ethyl ester;
4) 1-[(2S, 3S, 4R)-3,4-dihydro-2-dimethoxymethyl-3-hydroxy-2-methyl-6-nitro-2H-1-benzopyran-4-yl]-(2S)-2,3-dihydro-1H-indole-2-carboxylic acid ethyl ester;
5) 1-[(2S, 3R, 4S)-3,4-dihydro-2-dimethoxymethyl-3-hydroxy-2-methyl-6-nitro-2H-1-benzopyran-4-yl]-1H-(2R)-2,3-dihydroindole-2-carboxylic acid methyl ester;
6) 1-[(2S, 3R, 4S)-3,4-dihydro-2-dimethoxymethyl-3-hydroxy-2-methyl-6-nitro-2H-1-benzopyran-4-yl]-1H-(2S)-2,3-dihydroindole-2-carboxylic acid methyl ester;
7) 1-[(2S, 3S, 4R)-3,4-dihydro-2-dimethoxymethyl-3-hydroxy-2-methyl-6-nitro-2H-1-benzopyran-4-yl]-(2R)-2,3-dihydro-1H-indole-2-carboxylic acid methyl ester;
8) 1-[(2S, 3S, 4R)-3,4-dihydro-2-dimethoxymethyl-3-hydroxy-2-methyl-6-nitro-2H-1-benzopyran-4-yl]-(2S)-2,3-dihydro-1H-indole-2-carboxylic acid methyl ester;
9) 1-[(2S, 3R, 4S)-3,4-dihydro-2-dimethoxymethyl-3-hydroxy-2-methyl-6-nitro-2H-1-benzopyran-4-yl]-(2S)-2,3-dihydro-1H-indole-2-carboxylic acid;
10) 1-[(2S, 3R, 4S)-3,4-dihydro-2-dimethoxymethyl-3-hydroxy-2-methyl-6-nitro-2H-1-benzopyran-4-yl]-(2S)-2,3-dihydro-1H-indole-2-carboxylic acid isopropyl ester;
11) 1-[(2S, 3R, 4S)-3,4-dihydro-2-dimethoxymethyl-3-hydroxy-2-methyl-6-nitro-2H-1-benzopyran-4-yl]-(2R)-2,3-dihydro-1H-5-methoxyindole-2-carboxylic acid ethyl ester;
12) 1-[(2S, 3R, 4S)-3,4-dihydro-2-dimethoxymethyl-3-hydroxy-2-methyl-6-nitro-2H-1-benzopyran-4-yl]-(2S)-2,3-dihydro-1H-5-methoxyindole-2-carboxylic acid ethyl ester;
13) 1-[(2S, 3S, 4R)-3,4-dihydro-2-dimethixymethyl-3-hydroxy-2-methyl-6-nitro-2H-1-benzopyran-4-yl]-(2R)-2,3-dihydro-1H-5-methoxyindole-2-carboxylic acid ethyl ester;
14) 1-[(2S, 3S, 4R)-3,4-dihydro-2-dimethoxymethyl-3-hydroxy-2-methyl-6-nitro-2H-1-bezopyran-4-yl]-(2S)-2,3-dihydro-1H-5-methoxyindole-2-carboxylic acid ethyl ester;
15) 1-[(2S, 3R, 4S)-3,4-dihydro-2-dimethoxymethyl-3-hydroxy-2-methyl-6-nitro-2H-1-benzopyran-4-yl]-(2R)-2,3-dihydro-1H-5-fluoroindole-2-carboxylic acid ethyl ester;
16) 1-[(2S, 3R, 4S)-3,4-dihydro-2-dimethoxymethyl-3-hydroxy-2-methyl-6-nitro-2H-1-benzopyran-4-yl]-(2R)-2,3-dihydro-1H-5-fluoroindole-2-carboxylic acid ethyl ester;
17) 1-[(2S, 3R, 4S)-3,4-dihydro-2-dimethoxymethyl-3-hydroxy-2-methyl-6-nitro-2H-1-benzopyran-4-yl]-(2S)-2,3-dihydro-1H-5-fluoroindole-2-carboxylic acid ethyl ester;
18) 1-[(2S, 3S, 4R)-3,4-dihydro-2-dimethoxymethyl-3-hydroxy-2-methyl-6-nitro-2H-1-benzopyran-4-yl]-(2S)-2,3-dihydro-1H-5-fluoroindole-2-carboxylic acid ethyl ester;
19) 1-[(2S, 3R, 4S)-3,4-dihydro-2-dimethoxymethyl-3-hydroxy-2-methyl-6-nitro-2H-1-benzopyran-4-yl]-(2R)-2,3-dihydro-1H-5-chloroindole-2-carboxylic acid ethyl ester;
20) 1-[(2S, 3R, 4S)-3,4-dihydro-2-dimethoxymethyl-3-hydroxy-2-methyl-6-nitro-2H-1-benzopyran-4-yl]-(2S)-2,3-dihydro-1H-5-chloroindole-2-carboxylic acid ethyl ester;
21) 1-[(2S, 3S, 4R)-3,4-dihydro-2-dimethoxymethyl-3-hydroxy-2-methyl-6-nitro-2H-1-benzopyran-4-yl]-(2R)-2,3-dihydro-1H-5-chloroindole-2-carboxylic acid ethyl ester;
22) 1-[(2S, 3S, 4R)-3,4-dihydro-2-dimethoxymethyl-3-hydroxy-2-methyl-6-nitro-2H-1-benzopyran-4-yl]-(2S)-2,3-dihydro-1H-5-chloroindole-2-carboxylic acid ethyl ester;
23) 1-[(2S, 3S, 4R)-3,4-dihydro-2-dimethoxymethyl-3-hydroxy-2-methyl-6-nitro-2H-1-benzopyran-4-yl]-(2R)-2,3-dihydro-1H-5-chloroindole-2-carboxylic acid methyl ester;
24) 1-[(2S, 3S, 4S)-3,4-dihydro-2-dimethoxymethyl-3-hydroxy-2-methyl-6-nitro-2H-1-benzopyran-4-yl]-(2S)-2,3-dihydro-1H-5-chloroindole-2-carboxylic acid methyl ester;
25) 1-[(2S, 3R, 4S)-3,4-dihydro-2-dimethoxymethyl-3-hydroxy-2-methyl-6-nitro-2H-1-benzopyran-4-yl]-(2R)-2,3-dihydro-1H-5-chloroindole-2-carboxylic acid ethyl amide;
26) 1-[(2S, 3R, 4S)-3,4-dihydro-2-dimethoxymethyl-3-hydroxy-2-methyl-6-nitro-2H-1-benzopyran-4-yl]-(2S)-2,3-dihydro-1H-5-chloroindole-2-carboxylic acid ethyl amide;
27) 1[-(2S, 3R, 4S)-3,4-dihydro-2-dimethoxymethyl-3-hydroxy-2-methyl-6-nitro-2H-1-benzopyran-4-yl]-(2R)-2,3-dihydro-5-chloro-1H-2-hydroxymethylindole;
28) 1-[(2S, 3R, 4S)-3,4-dihydro-2-dimethoxymethyl-3-hydroxy-2-methyl-6-nitro-2H-1-benzopyran-4-yl]-(2S)-2,3-dihydro-5-chloro-1H-2-hydroxymethylindole;
29) 1-[(2R, 3S, 4R)-3,4-dihydro-2-dimethoxymethyl-3-hydroxy-2-methyl-6-nitro-2H-1-benzopyran-4-yl]-(2S)-2,3-dihydro-1H-indole-2-carboxylic acid ethyl ester;
30) 1-[(2R, 3S, 4R)-3,4-dihydro-2-dimethoxymethyl-3-hydroxy-2-methyl-6-nitro-2H-1-benzopyran-4-yl]-(2R)-2,3-dihydro-1H-indole-2-carboxylic acid ethyl ester;
31) 1-[(2R, 3R, 4S)-3,4-dihydro-2-dimethoxymethyl-3-hydroxy-2-methyl-6-nitro-2H-1-benzopyran-4-yl]-(2S)-2,3-dihydro-1H-indole-2-carboxylic acid ethyl ester;
32) 1-[(2R, 3R, 4S)-3,4-dihydro-2-dimethoxymethyl-3-hydroxy-2-methyl-6-nitro-2H-1-benzopyran-4-yl]-(2R)-2,3-dihydro-1H-indole-2-carboxylic acid ethyl ester;
33) 1-[(2R, 3S, 4R)-3,4-dihydro-2-dimethoxymethyl-3-hydroxy-2-methyl-6-nitro-2H-1-benzopyran-4-yl]-(2S)-2,3-dihydro-1H-indole-2-carboxylic acid n-propyl ester;
34) 1-[(2R, 3S, 4R)-3,4-dihydro-2-dimethoxymethyl-3-hydroxy-2-methyl-6-nitro-2H-1-benzopyran-4-yl]-(2R)-2,3-dihydro-1H-indole-2-carboxylic acid n-propyl ester;
35) 1-[(2R, 3S, 4R)-3,4-dihydro-2-dimethoxymethyl-3-hydroxy-2-methyl-6-nitro-2H-1-benzopyran-4-yl]-(2S)-2,3-dihydro-1H-5-methoxyindole-2-carboxylic acid ethyl ester;
36) 1-[(2R, 3S, 4R)-3,4-dihydro-2-dimethoxymethyl-3-hydroxy-2-methyl-6-nitro-2H-1-benzopyran-4-yl]-(2R)-2,3-dihydro-1H-5-methoxyindole-2-carboxylic acid ethyl ester;
37) 1-[(2R, 3R, 4S)-3,4-dihydro-2-dimethoxymethyl-3-hydroxy-2-methyl-6-nitro-2H-1-benzopyran-4-yl]-(2S)-2,3-dihydro-1H-5-methoxyindole-2-carboxylic acid ethyl ester;
38) 1-[(2R, 3R, 4S)-3,4-dihydro-2-dimethoxymethyl-3-hydroxy-2-methyl-6-nitro-2H-1-benzopyran-4-yl]-(2R)-2,3-dihydro-1H-5-methoxyindole-2-carboxylic acid ethyl ester;
39) 1-[(2R, 3S, 4R)-3,4-dihydro-2-dimethoxymethyl-3-hydroxy-2-methyl-6-nitro-2H-1-benzopyran-4-yl]-(2S )-2,3-dihydro-1H-5-methoxyindole-2-carboxylic acid ethyl amide;
40) 1-[(2R, 3S, 4R)-3,4-dihydro-2-dimethoxymethyl-3-hydroxy-2-methyl-6-nitro-2H-1-benzopyran-4-yl]-(2S)-2,3-dihydro-1H-5-chloroindole-2-carboxylic acid ethyl ester;
41) 1-[(2R, 3S, 4R)-3,4-dihydro-2-dimethoxymethyl-3-hydroxy-2-methyl-6-nitro-2H-1-benzopyran-4-yl]-1H-(2R)-2,3-dihydro-5-chloroindole-2-carboxylic acid ethyl ester;
42) 1-[(2S, 3R, 4S)-6-amino-3,4-dihydro-2-dimethoxymethyl-3-hydroxy-2-methyl -2H-1-benzopyran-4-yl]-(2R)-2,3-dihydro-1H-indole-2-carboxylic acid ethyl ester;
43) 1-[(2S, 3R, 4S)-6-amino-3,4-dihydro-2-dimethoxymethyl-3-hydroxy-2-methyl-2H-1-benzopyran-4-yl]-(2S)-2,3-dihydro-1H-indole-2-carboxylic acid ethyl ester;
44) 1-[(2S, 3S, 4R)-6-amino-3,4-dihydro-2-dimethoxymethyl-3-hydroxy-2-methyl-2H-1-benzopyran-4-yl]-(2R)-2,3-dihydro-1H-indole-2-carboxylic acid ethyl ester;
45) 1-[(2S, 3S, 4R)-6-amino-3,4-dihydro-2-dimethoxymethyl-3-hydroxy-2-methyl-2H-1-benzopyran-4-yl]-(2S)-2,3-dihydro-1H-indole-2-carboxylic acid ethyl ester;
46) 1-[(2S, 3R, 4S)-3,4-dihydro-2-dimethoxymethyl-3-hydroxy-2-methyl-6-nitro-2H-1-benzopyran-4-yl]-1H-indole-2-carboxylic acid ethyl ester;
47) 1-[(2S, 3S, 4R)-3,4-dihydro-2-dimethoxymethyl-3-hydroxy-2-methyl-6-nitro-2H-1-benzopyran-4-yl]-1H-indole-2-carboxylic acid ethyl ester;
48) 1-[(2S, 3R, 4S)-3,4-dihydro-2-dimethoxymethyl-3-hydroxy-2-methyl-6-nitro-2H-1-benzopyran-4-yl]-1H-indole-2-carboxylic acid methyl ester;
49) 1-[(2S, 3S, 4R)-3,4-dihydro-2-dimethoxymethyl-3-hydroxy-2-methyl-6-nitro-2H-1-benzopyran-4-yl]-1H-indole-2-carboxylic acid methyl ester;
50) 1-[(2S, 3R, 4S)-3,4-dihydro-2-dimethoxymethyl-3-hydroxy-2-methyl-6-nitro-2H-1-benzopyran-4-yl]-1H-indole-2-carboxylic acid;
51) 1-[(2S, 3S, 4R)-3,4-dihydro-2-dimethoxymethyl-3-hydroxy-2-methyl-6-nitro-2H-1-benzopyran-4-yl]-1H-indole-2-carboxylic acid;
52) 1-[(2S, 3R, 4S)-6-amino-3,4-dihydro-2-dimethoxymethyl-3-hydroxy-2-methyl-2H-1-benzopyran-4-yl]-1H-indole-2-carboxylic acid ethyl ester;
53) 1-[(2S, 3S, 4R)-6-amino-3,4-dihydro-2-dimethoxymethyl-3-hydroxy-2-methyl-2H-1-benzopyran-4-yl]-1H-indole-2-carboxylic acid ethyl ester;
54) 1-[(2S, 3R, 4S)-3,4-dihydro-2-dimethoxymethyl-3-hydroxy-2-methyl-6-nitro-2H-1-benzopyran-4-yl]-(2R)-1,2,3,4-tetrahydro-1H-quinoline-2-carboxylic acid methyl ester;
55) 1-[(2S, 3R, 4S)-3,4-dihydro-2-dimethoxymethyl-3-hydroxy-2-methyl-6-nitro-2H-1-benzopyran-4-yl]-(2S)-1,2,3,4-tetrahydro-1H-quinoline-2-carboxylic acid methyl ester;
56) 1-[(2S, 3S, 4R)-3,4-dihydro-2-dimethoxymethyl-3-hydroxy-2-methyl-6-nitro-2H-1-benzopyran-4-yl]-(2R)-1,2,3,4-tetrahydro-1H-quinoline-2-carboxylic acid methyl ester;
57) 1-[(2S, 3S, 4R)-3,4-dihydro-2-dimethoxymethyl-3-hydroxy-2-methyl-6-nitro-2H-1-benzopyran-4-yl]-(2S)-1,2,3,4-tetrahydro-1H-quinoline-2-carboxylic acid methyl ester;
58) (2S, 2aR, 4aR, 10aS)-[(3,4-b)-2a,10a-dihydro-2-dimethoxymehyl-2-methyl-12-nitro-2H-1-benzopyrano]-[(1,2-d)-4a,5-dihydro-10H-indolino]-4-oxomorpholine;
59) (2S, 2aR, 4aS, 10aS)-[(3,4-b)-2a,10a-dihydro-2-dimethoxymethyl-2-methyl-12-nitro-2H-1-benzopyrano]-[(1,2-d)-4a,5-dihydro-10H-indolino]-4-oxomorpholine;
60) (2S, 2aS, 4aR, 10aR)-[(3,4-b)-2a,10a-dihydro-2-dimethoxymethyl-2-methyl-12-nitro-2H-1-benzopyrano]-[(1,2-d)-4a,5-dihydro-10H-indolino]-4-oxomorpholine;
61) (2S, 2aS, 4aS, 10aR)-[(3,4-b)-2a,10a-dihydro-2-dimethoxymethyl-2-methyl-12-nitro-2H-1-benzopyrano]-[(1,2-d)-4a,5-dihydro-10H-indolino]-4-oxomorpholine;
62) (2S, 2aR, 10aS)-[(3,4-b)-2a,10a-dihydro-2-dimethoxymethyl-2-methyl-12-nitro-2H-1-benzopyrano]-[(1,2-d)-10H-indolino]-4-oxomorpholine; or
63) (2S, 2aS, 10aR)-[(3,4-b)-2a,10a-dihydro-2-dimethoxymethyl-2-methyl-12-nitro-2H-1-benzopyrano]-[(1,2-d)-10H-indolino]-4-oxomorpholine.
The more preferable compounds of the present invention are:
1-[(2S, 3R, 4S)-3,4-dihydro-2-dimethoxymethyl-3-hydroxy-2-methyl-6-nitro-2H-1-benzopyran-4-yl]-(2S)-2,3-dihydro-1H-indole-2-carboxylic acid ethyl ester;
1-[(2S, 3R, 4S)-3,4-dihydro-2-dimethoxymethyl-3-hydroxy-2-methyl-6-nitro-2H-1-benzopyran-4-yl]-(2R)-2,3-dihydro-1H-5-methoxyindole-2-carboxylic acid ethyl ester;
1-[(2S, 3R, 4S)-3,4-dihydro-2-dimethoxymethyl-3-hydroxy-2-methyl-6-nitro-2H-1-benzopyran-4-yl]-(2S)-2,3-dihydro-1H-5-chloroindole-2-carboxylic acid ethyl ester;
1-[(2R, 3S, 4R)-3,4-dihydro-2-dimethoxymethyl-3-hydroxy-2-methyl-6-nitro-2H-1-benzopyran-4-yl]-(2R)-2,3-dihydro-1H-indole-2-carboxylic acid ethyl ester;
1-[2R, 3S, 4R)-3,4-dihydro-2-dimethoxymethyl-3-hydroxy-2-methyl-6-nitro-2H-1-benzopyran-4-yl]-(2S)-2,3-dihydro-1H-5-methoxyindole-2-carboxylic acid ethyl ester;
1-[(2R, 3S, 4R)-3,4-dihydro-2-dimethoxymethyl-3-hydroxy-2-methyl-6-nitro-2H-1-benzopyran-4-yl]-(2R)-2,3-dihydro-1H-5-methoxyindole-2-carboxylic acid ethyl ester;
1-[(2R, 3S, 4R)-3,4-dihydro-2-dimethoxymethyl-3-hydroxy-2-methyl-6-nitro-2H-1-benzopyran-4-yl]-(2S)-2,3-dihydro-1H-5-chloroindole-2-carboxylic acid ethyl ester; and
1-[(2R, 3S, 4R)-3,4-dihydro-2-dimethoxymethyl-3-hydroxy-2-methyl-6-nitro-2H-1-benzopyran-4-yl]-1H-(2R)-2,3-dihydro-5-chloroindole-2-carboxylic acid ethyl ester.
The compounds of formula 1 may be used as pharmaceutically acceptable salts derived from pharmaceutically or physiologically acceptable free acids. These salts include but are not limited to the following: salts with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, sulfonic acid, phosphoric acid, stannic acid, etc. and organic acids such as citric acid, acetic acid, lactic acid, maleic acid, fumaric acid, gluconic acid, methanesulfonic acid, glycolic acid, succinic acid, tartaric acid, 4-toluenesulfonic acid, galacturonic acid, embonic acid, glutamic acid, aspartic acid, etc. The acid salts of the compounds according to the present invention can be prepared in the customary manner, for example by dissolving the compound of formula 1 in excess aqueous acid and precipitating the salt with a water-miscible organic solvent, such as methanol, ethanol, acetone or acetonitrile. It is also possible to prepare by heating equivalent amounts of the compound of formula 1 and an acid in water or an alcohol, such as glycol monomethyl ether, and then evaporating the mixture to dryness or filtering off the precipitated salt with suction.
Also the compounds of formula 1 may be in the form of pharmaceutically acceptable ammonium, alkali metals or alkaline earth metals salts. The alkali metal or alkaline earth metal salts of the compound of formula 1 can be obtained, for example, by dissolving the compound of formula 1 in exess alkali metal or alkaline earth metal hydroxide solution, filtering off the undissolved materials and evaporating the filtrate to dryness. Sodium, potassium or calcium salts are pharmaceutically suitable. The corresponding silver salts are obtained by the reaction of an alkali metal or alkaline earth metal salt with a suitable silver salt, such as silver nitrate.
In addition, the present invention provides processes for preparing of the benzopyranyl heterocycle derivatives of formula 1.
In particular, the present invention provides processes for preparing the benzopyranyl heterocycle derivatives of formula 1, represented by the following scheme 1. 
Wherein R1, R3, R4 and n are each defined as above.
In addition, the present invention provides processes for preparing the benzopyranyl heterocycle derivatives of formula 1 by using the compound (Ixe2x80x2) prepared in the scheme 1, represented by the following scheme 2. 
Wherein R1, R2, R3, R4 and n are each defined as above.
The substituents of R1, R2, R3 and R4 can be modified or 2,3-double bond in heterocycle can be formed via the reaction represented by the above scheme 2.
The derivatives of formula 1 can be prepared separately as an optically active isomer by using the corresponding optical isomer as a starting material.
In case of using a racemic mixture as a starting material, the derivatives of formula 1 are prepared as a racemic or a diastereomeric mixture, which can be separated into each optical isomers. The optical isomers can be separated by common chiral column chromatography or recrystallization.
The compounds of formula 1 can be synthesized using the reactions and techniques described herein below. The reactions are performed in a solvent appropriate to the reagents and materials employed and suitable for the transformation being effected.
I. Preparation of Starting Materials
Epoxy compounds (II) which were used as a starting material in scheme 1, can be prepared by the reaction represented by the following scheme 3. 
Wherein R1 is defined as above, (OZ) represents a leaving group and Hal represents a halogen atom.
The method for the preparation of the epoxide compound (II) represented by the above scheme 3 is described in U.S. Pat. No. 5,236,935 and KR Pat. No. 096,546 which were acquired by the present inventors, in detail.
(1) Preparation of Olefin Compounds (V)
Olefin compounds (V) exist as enantiomers (V1 and V2) such as formula 5. 
Wherein R1 is defined as above.
Olefin compound (V) can be prepared by the method disclosed in KR Pat. Appln. No. 96-7399 according to the present inventors. The following scheme 4 shows the detail process for the preparation of the olefin compound (V) from an alcohol compound (IV), prepared in scheme 3. The olefin compounds (V) can be obtained separately as an optically active olefin compound (V1) and olefin compound (V2) of formula 5, respectively. 
Wherein R1 is defined as above.
(2) Preparation of Epoxide Compounds (II)
Epoxide compounds (II1) and epoxide compounds (II2) can be prepared from the compound (V1) and epoxide compounds (II3) and epoxide compounds (II4) can be prepared from the compound (V2) as represented by the following scheme 5, by using the compound (V1) and the compound (V2) prepared in scheme 4 as a starting material, respectively. 
Wherein R1 is defined as above.
The epoxide compounds (II1) and (II2) can be separated to each optical isomer, and all the separated epoxide compounds or the mixture thereof can be used in the next step. Also the epoxide compounds (II3) and (II4) can be separated, and all the separated epoxide compounds or the mixture thereof can be used in the next step.
Epoxide compounds (II1) and (II2) and epoxide compounds (II3) and (II4) can be prepared from olefin compounds (V1) and (V2), respectively, by the preparation method disclosed in U.S. Pat No. 5,236,935 and KR Pat. No. 096,546 which were acquired by the present inventors.
Also, the epoxide compound (II) can be prepared from propazylether derivatives [J. Med. Chem. 26, 1582 (1983)].
It is also possible to prepare optical isomers (II1), (II2), (II3) and (II4) of epoxide compounds, respectively, from olefin compounds (V1) or (V2), by using Mn(III) salen epoxidation catalysts [E. N. Jacobsen et al., Tetrahedron Lett., 38, 5055 (1991)]. In case of using (R,R)xe2x80x94Mn(III) salen catalyst, epoxide compounds (II1) can be prepared from olefin compounds (V1) and epoxide compounds (II3) from the olefin compounds (V2). In case of using (S,S)xe2x80x94Mn(III) salen catalyst, epoxide compounds (II2) can be prepared from the olefin compounds (V1) and epoxide compounds (II4) from the olefin compounds (V2). This epoxidation reaction is performed in co-solvent of methylene chloride and water by using NaOCl as an oxidizing agent.
(3) Preparation of Heterocycle Comopunds (III)
Heterocyclic amine compounds (III) which were used as a starting material in the above scheme 1, can be prepared from the selective reduction of aromatic hetero-ring compounds (VI) represented by following scheme 6. 
Wherein R3, R4, and n are each defined as above.
Hetero-rings such as pyrrole and pyridine in heterocyclic compounds (VI) can be selectively reduced through the hydrogenation reaction using the metal catalysts such as platinum, Raney-nickle, etc. Preferred solvents are alcohols such as methanol, ethanol, etc.
In the above scheme 6, indole compounds in which n is 0, can be reduced to indoline by using NaCNBH3 as a reducing agent in trifluoroacetic acid. Reaction temperature may range from 0xc2x0 C. to rt. In addition, magnesium turning in methanol can reduce the pyrrole ring of indole compounds.
In the above scheme 6, quinoline compounds in which n is 1, can be reduced by using NaCNBH3 as a reducing agent in acidic pH, which has to be maintained as pH 4 using methanolic HCl in co-solvents of tetrahydrofuran, and methanol.
Amine compounds (III1) and (III2) can be separated to each optical isomer, and all the separated amine compounds or the mixture thereof can be used in next step. Optical isomers (III1) and (III2) can be separated by the reaction with enzymes such as hydrolases, or by the chromatography using chiral stationary column [W. H. Pirkle et al., J. of Chromatography, 316, 585 (1984)], or by the crystallization of salts with cinchonidine [J. L. Stanton et al., J. Med. Chem. 26, 1267 (1983)], etc., using different solubility between optical isomers.
In addition, optical isomer (III1) and (III2) can be synthesized separately by the chiral auxiliary mediated reduction of aromatic heterocycle compounds (VI) [A. V. Karchava et al., Tetrahedron: Asymmetry, 6, 2895 (1995)], by the chiral auxiliary mediated heterocyclic ring formation [J. P. Marino et al., J. Am. Chem. Soc. 114, 5566 (1992); A. I. Meyers et al., J. Org. Chem. 57, 3673 (1992)], or by the asymmetric substitution at 2-position of N-protected indoline compounds using chiral ligand [A. I. Meyers et al., J. Org. Chem. 58,6538 (1993); P. Beak et. Al., J. Org. Chem. 62, 7679 (1997)].
II. Preparation of Compound (IIxe2x80x2) from the Starting Compounds
The method for the preparation of compounds (formula 1) comprises the step of coupling an epoxide compound (II) and heterocyclic amine compound (III) in the presence of NaH, K2CO3, t-BuOK, Mg(ClO4)2, CoCl2, etc. The compound (Ixe2x80x2), which is a compound of formula 1 with R2=OH and 2,3-single bond in heterocycles, is prepared by this reaction.
In case of using bases, preferable reaction solvent is ether such as tetrahydrofuran or substituted amide such as N,N-dimethylformamide, and in case of using metal salts, preferable solvent is acetonitrile. Reaction temperature may range from rt to boiling point of employed solvent.
In case of using each stereoisomer of the epoxide compound (II) and heterocyclic amine compounds (III) as a starting material, the product with the same configuration to that of the starting material is obtained, respectively. That is, the compounds (I8) (I6), (I1) or (I3) of formula 1 can be prepared from epoxide compounds (II1), (II2), (II3) or (II4) with amine compounds (III1), respectively. And the compounds (I7), (I5), (I2) or (I4) of formula 4 can be prepared from epoxide compounds (II1), (II2), (II3) or (II4) with amine compounds (III2), respectively. In case of using a stereoisomer of the epoxide compound (II1), (II2), (II3) or (II4), and a mixture of heterocyclic amine compounds (III) as a starting material, diastereomeric mixture of compounds (I8) and (I7), (I6) and (I5), (I1) and (I2), or (I3) and (I4) are obtained, respectively, which are separated by chiral column chromatography to give each stereoisomers.
III. Preparation of Compounds (I) from the Compounds (Ixe2x80x2)
For preparation of compound (I), the substituents R1, R2, R3 and R4 of the compound (Ixe2x80x2) can be modified to other functional groups, and a double bond can be introduced at 2,3-position of heterocycle by the reaction of scheme 2.
A starting material, reactants and the reaction condition are determined according to the structure of product, that is what are the substituents R1, R2, R3, and R4 and whether there is a double bond at 2,3-position of heterocycle. Therefore the present invention includes all the reaction types, reactants and reaction conditions by which it is possible to prepare the compound of formula 1.
Several processes for the preparation of the compounds of formula 1 according to scheme 2 are described below in detail. However, the description of the processes, reactants and reaction conditions should not be understood to limit the present invention.
(1) Introduction of Double Bond at 2, 3-position of Heterocycle
Benzopyranyl indole compounds (Ib) can be prepared from the coupling of epoxide compound (II) with indole amines (IIIb) as represented in the below scheme 7, but whose yields are very low already reported [K. S. A. Atwal et al., J. Med. Chem., 38, 3236 (1995)]. 
Wherein R1, R2, R3 and R4 are each defined as above.
Indoline compounds (Ia), of which n is 0, can be aromatized to indole compounds (Ib), which has a double bond at 2,3-position by oxidation as represented in the below scheme 8. Then, Aromitization of indoline compounds (around 80% yield) is more preferable to prepare benzopyranyl indole compounds (Ib). 
Wherein R1, R2, R3 and R4 are each defined as above.
The aromatization of indoline to indole can be carried out by using a oxidizing agent such as MnO2, DDQ (2,3-dichloro-5,6-dicyano-1,4-benzoquinone), etc. In the case of using DDQ, the reaction is proceeded at rt, and preferable solvents are aromatic solvents such as benzene, toluene, etc., and ethers such as dioxane, etc.
(2) Introduction of NH2 group at R1 
The compound (Ic) of formula 1 whose R1 is NH2 can be prepared by the reduction of the compound (Id) with R1=NO2 as represented in the below scheme 9. 
Wherein R2, R3, R4 and n are each defined as above.
The NO2 group can be reduced to NH2 group by hydrogenation using metal catalysts such as platinum, palladium, palladium on carbon (Pd/C), Raney-nickel, etc. in a suitable solvent. Preferred solvents are alcohols such as methanol, ethanol, etc., and ethyl acetate.
In addition, the reduction of NO2 group to NH2 group can be carried out by using a reducing agent such as NaBH4 in the presence of CuSO4, Cu(OAc)2, CoCl2, SnCl2 or NiCl2. At this time, preferred solvent is a mixture of water and methanol, and room temperature for reaction temperature is preferred.
(3) Lactone Ring Formation by the Intramolecular Esterification
The lactone compound (If) of formula 1 can be prepared by the intramolecular esterification of the compound (Ie) with R3=C(xe2x95x90O)ORa as represented in the below scheme 10. 
Wherein R2, R4, Ra and n are each defined as above.
In the case of using the carboxylic acid compound with Ra=H as a starting material, the lactone compound (If) can be prepared by activating to mixed anhydride with alkyl formate or to azide with diphenylphosphoryl azide, etc., or by condensing using N,Nxe2x80x2-dicyclohexylcarbodiimide (DCC), water-soluble carbodiimidazole (WSC), etc. Preferable solvents are ether type such as tetrahydrofuran, etc., and substituted amide such as N,N-dimethylformamide, etc.
In the case of using the carboxylic ester compound with Ra=alkyl or aryl as a starting material, the lactone compound (If) can be prepared using Lewis acid such as diethylchloro aluminum etc., in the presence of base catalyst such as diisopropylamine, etc. Preferable solvent is CH2Cl1, etc.
(4) Introduction of 
to R3 
The amide compound (Ig) whose R3 is 
can be prepared by the aminolysis of the compound (Ie) with R3 =C(xe2x95x90O)ORa or by the coupling of the heterocyclic amide compound (IIIg) with epoxide (II), as represented in the below scheme 11. 
Wherein R1, R2, R4, Ra and n are each defined as above.
Preferable solvent for aminolysis is alcohol such as methanol, ethanol, etc.
The molecular structure of the compounds according to the present invention was identified by IR spectroscopy, UV spectroscopy, NMR spectroscopy, mass spectroscopy, liquid chromatography, X-ray diffraction, optical rotation analysis and elemental analysis.
In addition, the present invention provides pharmaceutical compositions which contain the benzopyranyl heterocycle derivatives of the above formula 1 or their pharmaceutically acceptable salts as an active ingredient. In particular, the present invention provides pharmaceutical compositions for protecting heart, protecting neuronal cells, protecting from brain injury, or suppressing lipid peroxidation.
In the experiments using isolated rat aorta, the compounds of the present invention showed remarkably low vasorelaxant activity compared to the reference KATP openers such as Cromakalim and BMS-180448. The KATP openers usually have both cardioprotective and vasodilating properties, and those are reported not to have correlation between them [K. S. Atwal et al., J. Med. Chem. 39, 304 (1996)]. The vasodilation effect is unnecessary, probably contraindicated for ischemia, due to underperfusion of the tissue already at risk. In other words, the vasorelaxant effect of these compounds would limit their utility in treating myocardial ischemia. As mentioned above, the compounds of the present invention are nearly devoid of vasorelaxant activity, thus their cardiac selectivity might offer a higher margin of safety as cardioprotectants.
Accordingly, the compounds of the present invention are confirmed their antiischemic activity with significant improvement in cardiac selectivity. In isolated ischemic rat heart model using Langendorff apparatus, the compounds of the present invention significantly prolong the time to contracture (TTC), improve the recovery of postischemic contractile function, and decrease the release of lactate dehydrogenase (LDH) which is a marker enzyme for cell injury. In the ischemic myocardium injury models of anesthetized rats, the compounds of the present invention exhibited equal or superior antiischemic activity compared to that of BMS-180448. Further, in contrast to BMS-180448, the compounds of the present invention have noticeably low vasorelaxant activity and thus, they are superior to the conventional drugs as cardiac selective cardioprotectants.
As described above, the compounds of the present invention exert excellent anti-ischemic activity both in vitro and in vivo, while show low vasorelaxant acitivity, so that they can be used for the prevention or treatment of the diseases related to myocardial ischemia, such as postischemic contractile dysfunction, myocardial cell injury, and change of energy metabolism as well as a cardioprotective.
In addition, the compounds of the present invention have an ability to protect neurons. In detail, the compounds of the present invention protect neurons from oxidative stress by iron. Therefore, the compounds of the present invention can be used as a neuroprotective and can also be applied for the treatment of neurodegenerative disorders caused by the apoptosis or necrosis of neurons, such as stroke and cerebral dementia.
Further, the compounds of the present invention inhibit the lipid peroxidation induced by iron. Hence, the compounds of the present invention can be used as an antioxidant against lipid peroxidation and can be effectively applied for the medical treatment of the neurological disorders caused by the accumulation of free radical species within neurons, such as a stroke and dementia.
The present invention includes pharmaceutical formulations which contain, in addition to non-toxic, inert pharmaceutically suitable additives, one or more than one active ingredients according to the present invention and processes for the preparation of these formulations.
Non-toxic inert pharmaceutically suitable vehicle include solid, semi-solid or liquid diluents, fillers and formulation additives of all types.
Preferred pharmaceutical formulations are tablets, coated tablets, capsules, pills, granules, suppositories, solutions, suspensions and emulsions, pastes, ointments, gels, creams, lotions, dusting powders and sprays.
Tablets, coated tablets, capsules, pills and granules can contain the more than one additives in addition to the active ingredient or ingredients, such as (a) fillers and diluents, for example starches, lactose, sucrose, glucose, mannitol and silicic acid, (b) binders, for example carboxymethylcellulose, alginates, gelatine and polyvinylpyrrolidone, (c) humectants, for example glycerol, (d) disintegrants, for example agar-agar, calcium carbonate and sodium carbonate, (e) solution retarders, for example paraffin, and (f) absorption accelerators, for example quaternary ammonium compounds, (g) wetting agents, for example cetyl alcohol and glycerol monostearate, (h) adsorbents, for example kaolin and bentonite, and (i) lubricants, for example talc, calcium stearate, magnesium stearate, and solid polyethylene glycols, or mixtures of the substances listed under (a) to (i).
The tablets, coated tablets, capsules, pills and granules can be provided with the customary coatings and shells, optionally containing opacifying agents, and can also be of a composition such that they release the active ingredient or ingredients only or preferentially in a certain part of the intestinal tract, if appropriate in a delayed manner, examples of embedding compositions which can be used being polymeric substances and waxes.
If appropriate, the active ingredient or ingredients can also be present in microencapsulated form with one or more of the above mentioned excipients.
Suppositories can contain, in addition to the active ingredient or ingredients, the customary water-soluble or water-insoluble excipients, for example polyethylene glycols, fats, for example cacao fat, and higher esters (for example, C14-alcohol with C16-fatty acid) or mixtures of these substances.
Ointments, pastes, creams and gels can contain, in addition to the active ingredient or ingredients, the customary excipients, for example animal and vegetable fats, waxes, paraffins, starch, tragacanth, cellulose derivatives, polyethylene glycols, silicones, bentonites, silicic acid, talc and zinc oxide, or mixtures of these substances.
Dusting powders and sprays can contain, in addition to the active ingredient or ingredients, the customary excipients, for example lactose, talc, silicic acid, aluminum hydroxide, calcium silicate and polyamide powder, or mixtures of these substances. Sprays can additionally contain the customary propellants, for example chlorofluorohydrocarbons.
Solutions and emulsions can contain, in addition to the active ingredient or ingredients, the customary excipients, such as solvents, solubilizing agents and emulsifiers, for example water, ethyl alcohol, isopropyl alcohol, ethylcarbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, dimethylformamide, oils, in particular cottonseed oil, groundnut oil, corn germ oil, olive oil, castor oil and sesame oil, glycerol, glycerol formal, tetrahydrofurfuyl alcohol, polyethylene glycols and fatty acid esters of sorbitan, or mixtures of these substances.
For parenteral administration, the solutions and emulsions are also be in a sterile form which is isotonic with blood.
Suspensions can contain, in addition to the active ingredient or ingredients, the customary excipients, such as liquid diluents, for example water, ethyl alcohol and propylene glycol, and suspending agents, for example ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth, or mixtures of these substances.
The formulation forms mentioned can also contain coloring agents, preservatives and additives which improve the smell and taste, for example peppermint oil and eucalyptus oil, and sweeteners, for example saccharin.
The therapeutically active ingredients should preferably be present in the abovementioned pharmaceutical formulations in a concentration of about 0.1 to 99.5, preferably about 0.5 to 95% by weight of the total mixture.
The abovementioned pharmaceutical formulations can also contain other pharmaceutical active compounds in addition to the compounds according to the present invention.
The abovementioned pharmaceutical formulations are prepared in the customary manner by known methods, for example by mixing the active ingredient or ingredients with vehicles.
The formulations mentioned can be used on humans and animals either orally, rectally, parenterally (intravenously, intramuscularly or subcutaneously), intracisternally, intravaginally, intraperitoneally or locally (dusting powder, ointment, drops) and for the therapy of infections in hollow spaces and body cavities. Possible suitable formulations are injection solutions, solutions and suspensions for oral therapy and gels, infusion formulations, emulsions, ointments or drops, ophthalmological and dermatological formulations, silver salts and other salts, eardrops, eye onintments, dusting powders or solutions can be used for local therapy. In the case of animals, intake can also be in suitable formulations via the feed or drinking water.
Gels, powders, dusting powders, tablets, delayed release tablets, premixes, concentrates, granules, pellets, capsules, aerosols, sprays and inhalants can furthermore be used on humans and animals. The compounds according to the present invention can moreover be incorporated into other carrier materials, such as for example, plastics (chain of plastic for local therapy), collagen or bone cement.
In general, it has proved advantageous both in human medicine to administer the active compound or compounds according to the present invention in total amounts of about 0.1 to about 100, preferably 0.1 to 20 mg/kg of body weight every 24 hours, if appropriate in the form of several individual doses, to achieve the desired results. However, it may be necessary to deviate from the dosages mentioned, and in particular to do so as a function of the nature and body weight of the object to be treated, the nature and severity of the disease, the nature of the formulation and of the administration of the medicament and the period or interval within which administration takes place.
Thus in some cases it can suffice to manage with less than the abovementioned amount of active ingredient, while in other cases the abovementioned amount of active ingredient must be exceeded. The particular optimum dosage and mode of administration required for the active ingredient can be determined by any expert on the basis of his expert knowledge.
The starting materials, (II) and (III) of scheme 1 or scheme 2 were prepared from the following preparation examples.
To the pre-cooled solution of 0.55 M NaOCl (110 mL, 60.0 mmol) and 0.05 M Na2HPO4 (43 mL) at 0xc2x0 C., was added (2S)-2-dimethoxymethyl-2-methyl-6-nitro-2H-1-benzopyran (4 g, 15 mmol) and (R,R) Jacobson""s catalyst (477.5 mg, 0.75 mmol) in CH2Cl2 (20 mL). The reaction mixture was stirred at rt for 8 hr and then filtered through Celite to remove the jacobson""s catalyst. The layer was separated and the organic layer was washed with brine, dried (Na2SO4), filtered, then concentrated under reduced pressure. The residue was purified by silicagel column chromatography (n-hexane:ethyl acetate=4:1) to give the desired compound (3.24 g, 77%) as a light yellow solid.
1H NMR (CDCl3, 200 MHz) xcex41.28 (s, 3H), 3.60 (s, 3H), 3.67 (s, 3H), 3.80 (d, 1H), 3.96 (d, 1H), 4.47 (s, 1H), 6.94 (d, 1H), 8.15 (dd, 1H), 8.30 (d, 1H)
The reaction was proceeded by the same method used for the preparation example 1 above, except using (S,S) Jacobson""s catayst. The residue was purified by column chromatography (n-hexane:ethyl acetate=4:1) to give the desired compound (3.81 g, 90%) as a light yellow solid.
1H NMR (CDCl3, 200 MHz) xcex41.56 (s, 3H), 3.28 (s, 3H), 3.49 (s, 3H), 3.82 (d, 1H), 4.11 (d, 1H), 4.22 (s, 1H), 6.85 (d, 1H), 8.13 (dd, 1H), 8.27 (d, 1H)
To the pre-cooled solution of 0.55 M NaOCl (55 mL, 30.0 mmol) and of 0.05 M Na2HPO4 (21.5 mL) at 0xc2x0 C., (2R)-2-dimethoxymethyl-2-methyl-6-nitro-2H-1-benzopyran n(2 g, 7.5 mmol) and (R, R) Jacobson""s catalyst (477.5 mg, 0.75 mmol) in CH2Cl2 (10 mL) was added. The reaction mixture was stirred at rt for 8 hr and then filtered through Celite to remove the jacobson""s catalyst. The layer was separated and the organic layer was washed with brine, dried (Na2SO4), filtered, then concentrated under reduced pressure. The residue was purified by silicagel column chromatography (n-hexane:ethyl acetate=4:1) to give the desired compound (1.64 g, 77%) as a light yellow solid.
1H NMR (CDCl3, 200 MHz) xcex41.28 (s, 3H), 3.60 (s, 3H), 3.67 (s, 3H), 3.80 (d, 1H), 3.96 (d, 1H), 4.47 (s, 1H), 6.94 (d, 1H), 8.15 (dd, 1H), 8.30 (d, 1H)
The reaction was proceeded by the same method used for the preparation example 3 above, except using (R,R) Jacobson""s catalyst. The residue was purified by column chromatography (n-hexane:ethyl acetate=4:1) to give the desired product (1.55 g, 78%) as a light yellow solid.
1H NMR (CDCl3, 200 MHz) xcex41.56 (s, 3H), 3.28 (s, 3H), 3.49 (s, 3H) 3.82 (d, 1H), 4.11 (d, 1H), 4.22 (s, 1H) 6.85 (d, 1H), 8.13 (dd, 1H), 8.27 (d, 1H)
To cooled trifluoroacetic acid (40 mL) at 0xc2x0 C., NaCNBH3 (1.57 g, 25 mmol) was added portionwise with stirring. The reaction mixture was stirred for 15 min, and to which indole-2-carboxylic acid ethyl ester (1.2 g, 6.35 mmol) was added slowly, then the mixture was stirred at rt for an hour. After the completion of the reaction, water (150 mL) was added to the mixture, and which was stirred for 5 hr. The reaction was extracted by CH2Cl2 (40 mLxc3x973), then organic layer was washed with saturated aqueous solution of NaHCO3 (40 mLxc3x972) and water (40 mL), dried (Na2SO4), filtered, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (n-hexane:ethyl acetate=9:1) to give the desired compound (1.09 g, 90%).
1H NMR (CDCl3, 200 MHz) xcex41.29 (t, 3H), 3.34 (m, 2H), 4.07 (bs, 1H), 4.20 (q, 2H), 4.36 (dd, 1H), 6.74 (m, 2H), 7.08 (m, 2H)
(Step 1) Preparation of 5-fluoroindole-2-carboxylic acid ethyl ester
To the solution of 5-fluoroindole-2-carboxylic acid (1.0 g, 5.58 mmol) in DMF (10,0 mL), was added K2CO3 (1.0 g, 7.80 mmol), followed by bromoethane (685 mg, 6.29 mmol) dropwise at 0xc2x0 C. After the reaction mixture was stirred at rt for 10 hr, water (30 mL) was added to the reaction mixture, which was extracted with ethyl acetate (15 mLxc3x973). The organic layer was washed with brine and water, dried (Na2SO4), filtered and concentrated under reduced pressure to give the desired product (820 mg, 71%), which was used for the next step without further purification.
1H NMR (CDCl3, 200 MHz) xcex41.42 (t, 3H), 4.43 (q, 2H), 7.08 (ddd, 1H), 7.18 (s, 1H), 7.38 (m, 2H), 9.34 (bs, 1H)
(step 2) Preparation of 2,3-Dihydro-1H -5-fluoroindole-2-carboxylic acid ethyl ester
To cooled trifluoroacetic acid (15 mL) at 0xc2x0 C., NaCNBH3 (730 mg, 11.6 mmol) was added portionwise with stirring. The reaction mixture was stirred for 15 min, and to which the compound obtained from step 1 (600 mg, 2.90 mmol) was added slowly, then the mixture was stirred at rt for an hour. After the completion of reaction, water (150 mL) was added to the mixture, which was stirred for 5 hr. The reaction was extracted by CH2Cl2 (20 mLxc3x973), then organic layer was washed with saturated aqueous solution of NaHCO3 (20 mLxc3x972) and water (20 mL), dried (Na2SO4), filtered, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (n-hexane:ethyl acetate=9:1) to give the desired compound (605 mg, 63%)
1H NMR (CDCl3, 200 MHz) xcex41.29 (t, 3H), 3.32 (m, 2H), 4.25 (q, 2H), 4.39 (m, 1H), 6.62 (ddd, 1H), 6.79 (m, 2H)
To cooled trifluoroacetic acid (15 mL) at 0xc2x0 C., NaCNBH3 (1.50 mg, 23.87 mmol) was added portionwise with stirring. The reaction mixture was stirred for 15 min, and to which 5-chloroindole-2-carboxylic acid ethyl ester (1.0 g, 4.47 mmol) was added slowly, then the mixture was stirred at rt for an hour. After the completion of reaction, water (150 mL) was added to the mixture, which was stirred for 5 hr. The reaction was extracted by CH2Cl2 (40 mLxc3x973), then organic layer was washed with saturated aqueous solution of NaHCO3 (40 mLxc3x972) and water (40 mL), dried (Na2SO4), filtered, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (n-hexane:ethyl acetate=9:1) to give the desired compound (870 mg, 87%)
1H NMR (CDCl3, 200 MHz) xcex41.29 (t, 3H), 3.35 (m, 2H), 4.24 (q, 2H), 4.37 (dd, 1H), 6.62 (d, 1H), 7.03 (m, 2H)
(Step 1) Preparation of 1H-5-methoxyindole-2-carboxylic acid ethyl ester
Except using 5-methoxyindole-2-carboxylic acid (1.0 g, 5.23 mmol) as a starting material, the reaction was proceeded by the same method used for the step 1 of preparation example 6 above, which gave the desired product (985 mg, 86%).
1H NMR (CDCl3, 200 MHz) xcex41.41 (t, 3H), 3.85 (s, 3H), 4.40 (q, 2H), 7.01 (dd, 1H), 7.07 (s, 1H), 7.14 (d, 1H), 7.29 (d, 1H), 8.82 (bs, 1H)
(Step 2) Preparation of 2,3-dihydro-1H-5-methoxyindole-2-carboxylic acid ethyl ester
Except using the compound (985 mg, 4.50 mmol) prepared from step 1 as a starting material, the reaction was proceeded by the same procedure used for the step 2 of preparation example 6 above. The residue was purified by silica gel column chromatography (n-hexane:ethyl acetate=9:1) to give the desired compound (624 mg, 63%).
1H NMR (CDCl3, 200 MHz) xcex41.28 (t, 3H), 3.31 (m, 2H), 3.73 (s, 3H), 4.20 (q, 2H), 4.36 (dd, 1H),6.63 6.72 (m, 3H)
To the solution of indole-2-carboxylic acid ethyl ester (1.4 g, 8 mmol) in anhydrous methanol (40 mL), magnesium turnings (1.84 g, 80 mmol) were added portionwise with stirring. The reaction mixture was continuously stirred at 10-15xc2x0 C. until magnesium was completely dissolved, then poured onto the precooled an aqueous solution of 2N HCl at 4xc2x0 C. The reaction was basified to pH 9 with ammonia water, and extracted with ethyl acetate (20 mLxc3x973). The organic layer was washed with water (40 mL), dried (Na2SO4), dried, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (n-hexane:ethyl acetate=9:1) to give the desired compound (1.20 g, 85%).
1H NMR (CDCl3, 200 MHz) xcex43.34 (m, 2H), 3.74 (s, 3H), 4.21 (m, 1H), 6.75 (m, 2H), 7.06 (m, 2H)
Except using 5-chloroindole-2-carboxylic acid ethyl ester (3.0 g, 13.4 mmol) as a starting material, the reaction was proceeded by the same method used for the preparation example 9 above. The residue was purified by silica gel column chromatography (n-hexane:ethyl acetate=9:1) to give the desired compound (2.21 g, 78%).
1H NMR (CDCl3, 200 MHz) xcex43.36 (m, 2H), 3.79 (s, 3H), 4.43 (m, 1H), 6.64 (d, 1H), 7.04 (d, 2H)
(Step 1) Preparation of 2,3-Dihydro-1H-5-chloroindole-2-carboxylic acid
The compound (1.2 g, 5.67 mmol) obtained from preparation example 10 above, was dissolved in methanol (15 mL), to which was added KOH (57 mg, 6.14 mmol) in water (0.3 mL) . The reaction mixture was stirred at rt for 3 hr, then all volatiles were removed under reduced pressure. Water (10 mL) was added to the residue, which was acidified to pH 3 with c-HCl, and extracted with ethyl acetate (10 mLxc3x973). The extracts were dried (Na2SO4), filters and concentrated under reduced pressure to give the desired compound (1.01 g, 90%), which was used for the next step without further purification.
1H NMR (CD3OH, 200 MHz) xcex43.34 (m, 2H), 4.38 (dd, 1H), 6.61 (d, 1H), 6.93 (dd, 1H), 7.02 (d, 1H)
(Step 2) Preparation of 2,3-Dihydro-1H-5-chloro-2-hydroxymethylindole
To the precooled solution of the compound (198 mg, 1.0 mmol) obtained from step 1 in dry THF (2 mL) at 0xc2x0 C., 1N BH3 in THF (2.5 mL) was added dropwise via a syringe. The reaction mixture was stirred at rt for 5 hr, then concentrated in vacuo. To the residue water (5 mL) was added, which was extracted with ethyl acetate (5 mLxc3x973). The extracts were washed with saturated aqueous solution of NaHCO3, brine, and water, dried (Na2SO4), filtered, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (n-hexane:ethyl acetate=4:1) to give the desired compound (167 mg, 91%).
1H NMR (CDCl3, 200 MHz) xcex42.82 (dd, 1H), 3.11 (dd, 1H), 3.64 (m, 2H), 4.04 (m, 1H), 6.55 (d, 1H), 7.02 (m, 2H)
To the solution of (2S)-2,3-dihydroindole-2-carboxylic acid (163 mg, 1.0 mmol) in isopropyl alcohol (40 mL), was added thionyl chloride (0.1 mL) slowly. The reaction mixture was heated at reflux for 4 hr with stirring. After cooling, all volatiles were removed under reduced pressure. To the residue saturated aqueous solution of NaHCO3 (10 mL) was added, which was extracted with ethyl acetate (10 mLxc3x973). The extracts were dried (Na2SO4), filtered, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (n-hexane:ethyl acetate=4:1) to give the desired compound (182 mg, 89%).
1H NMR (CDCl3, 200 MHz) xcex41.25 (s, 3H), 1.28 (s, 3H), 3.28 (m, 2H), 4.48 (dd, 2H), 5.05 (q, 1H), 6.89 (m, 2H), 7.10 (m, 2H)
(Step 1) Preparation of 1H-quinoline-2-carboxylic acid methyl ester
To the solution of quinoline-2-carboxylic acid (500 mg, 2.89 mmol) in DMF (5 mL), K2CO3 (600 mg, 4.33 mmol) and iodomethane (412 mg, 2.9 mmol) were added at 0xc2x0 C., and the reaction mixture was stirred at rt for 3 hr. Water (20 mL) was added to the mixture, which was extracted with ethyl acetate (10 mLxc3x973). The extracts were dried (Na2SO4), filtered and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (n-hexane:ethyl acetate 9:1) to give the desired compound (414 mg, 77%).
1H NMR (CDCl3, 200 MHz) xcex44.08 (s, 3H), 7.61-7.92 (m, 3H), 8.18-8.38 (m, 3H)
(Step 2) Preparation of 1,2,3,4-tetrahydro-1H-quinoline-2-carboxylic acid methyl ester
To the solution of the compound (540 mg, 2.9 mmol) obtained from step 1 in THF (10 mL) and methanol (5 mL), were added NaCNBH3 (750 mg, 12 mmol) and bromocresol as an indicator, followed by methanolic HCl until yellow color was persistent. The reaction mixture was stirred at rt for 4 hr, then poured onto iced-water, and basified with NaHCO3, which was extracted with ethyl acetate (10 mLxc3x973). The extracts were dried (Na2SO4), filtered, concentrated under reduced pressure. The residue was purified by silica gel column chromatography (n-hexane:ethyl acetate=9:1) to give the desired compound (484 mg, 88%).
1H NMR (CDCl3, 200 MHz) xcex42.03 (m, 1H), 2.27 (m, 1H), 2.79 (m, 2H), 3.78 (s, 3H), 4.06 (dd,1H), 6.63 (dd, 2H), 6.97 (dd, 2H)